Liquid chromatography-mass spectrometry (LC/MS) is an extremely useful technique for detection, identification and (or) quantification of components of mixtures or of analytes within mixtures. As is known, liquid chromatography is a fractionation separation process. Accordingly, a liquid chromatograph instrument generally operates so as to separate a sample that is a complex mixture of substances into separate fractions. The individual fractions have simpler compositions than the original sample and the composition of each fraction can (but may not) approach that of a purified substance. The fraction compositions systematically vary from one another according to a gradient. The LC/MS technique generally provides data in the form of a mass chromatogram, in which detected ion intensity (a measure of the number of detected ions) as measured by a mass spectrometer is given as a function of time. In the LC/MS technique, various separated chemical constituents elute from a chromatographic column as a function of time. As these constituents elute off the column, they are submitted for mass analysis by a mass spectrometer at which each analyte or chromatographic fraction is ionized, generally producing a variety of ions from each such analyte or fraction. The mass spectrometer accordingly generates, in real time, detected relative ion abundance data for ions produced from each eluting analyte or each chromatographic fraction, in turn.
The term “liquid chromatography” includes, without limitation, reverse phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), high performance liquid chromatography (HPLC), ultra high performance liquid chromatography (UHPLC), normal-phase high performance liquid chromatography (NP-HPLC), supercritical fluid chromatography (SFC) and ion chromatography.
One can often enhance the specificity of the mass spectrometry technique by employing “tandem mass spectrometry” or “MS/MS”, for example via use of a triple quadrupole mass spectrometer. In the MS/MS technique, a parent (or precursor) ion generated from a molecule of interest can be filtered or isolated in an MS instrument (for instance, in a quadrupole mass filter, Q1, of a triple quadrupole instrument), and these precursor ions are subsequently fragmented (e.g., in a second quadrupole, Q2) to yield multiple product or fragment ions that are then analyzed in a downstream MS stage (e.g., in a third quadrupole, Q3). By careful selection of precursor ion and product ion species, the presence and/or concentrations of various analytes of interest can be determined with specificity. Multiple reaction monitoring (MRM) is performed by applying the above-described MS/MS procedure to multiple precursor/product ion pairs. When applied to the analysis of complex samples that are resolved using a liquid chromatograph (LC) to produce multiple constituents, the MRM technique provides sufficient throughput to screen or quantify a large fraction of the eluting analytes with high sensitivity and specificity.
An LC/MS analysis workflow that employs the MRM technique may be referred to as an LC-MRM analysis. During an LC-MRM analysis, the mass analyzer continuously cycles through all m/z ions included in a predetermined list for Q1 isolation over the duration of an LC gradient. The precursor m/z isolation (Q1 stage) only requires a few tens of milliseconds to isolate or filter ions comprising a single m/z range, thus permitting analysis of up to ˜500 isolations per second. For example, consider the model case depicted in FIG. 8 in which a chromatographic peak having an approximately 12 second width at its base is mass analyzed by the MRM technique and the MRM precursor isolation list includes 100 m/z species to be targeted during an LC analysis. The 100 m/z isolations are represented by square blocks at the base of FIG. 8. Each of the vertical lines illustrated underneath the chromatographic peak profile 200 represents a single, representative first mass spectral MS/MS analysis out of 100 such analyses per cycle. The exemplary absolute time scale shown at the base of FIG. 8 as well as in FIGS. 10A and 10B, as well as the implied absolute time scales illustrated in FIGS. 11 and 12 should be understood as being referenced to an analysis start time which is taken as the origin of the time axis (i.e., is taken as “time zero” at which t=0. The analysis start may be chosen to correspond to some well-defined event, such as the opening of a valve that begins the flow of chromatographic mobile phase through a chromatographic column. A consistent definition of the starting event enables comparison of results across separate experiments.
The time increment between each pair of vertical lines in the top portion of FIG. 8 represents the cumulative time needed to step through all the 100 MS/MS analyses—each corresponding to a different respective m/z isolation—and is termed the cycle time. The process is iterative; thus, a new sweep through the full list is initiated at the time indicated by each vertical line. This iterative analytical process terminates at the end of an LC gradient. LC-MRM analysis is a popular technique for quantifying constituents—such as proteins and peptides in biological samples—whose abundances may vary by orders of magnitude. At low abundance, the quality of quantitation is dependent on ion statistics or by the % RSD of the integral of the analyte response. An analytically acceptable % RSD of <15% often requires at least 10 MS/MS analyses. For instance, assume that the Q1 dwell time required to perform a single MS/MS analysis is equal to 10 ms and that the inter-analysis delay is 2.0 ms. These instrumental parameters correspond to a cycle time of ˜1.2 seconds per cycle, which is the time required to cycle through the 100 precursor/product ion pairs in the MRM list. Thus, during the elution of a ˜12-second wide peak, which is typical for nano-flow rate chromatography, a total of 10 mass spectral analyses can be acquired for each such ion pair.
A key difference between a MRM analysis and other types of tandem LC/MS analyses is that, in a MRM analysis, the detection of a precursor ion m/z is not a criterion to initiate a MRM event. The mass analyzer continuously cycles through a predetermined list of precursor-product ion pairs over the duration of a LC gradient. Conversely, in data dependent tandem mass analysis, a precursor ion species of interest must be detected in a low collision energy pre-scan or MS survey scan. The survey scan reveals high-abundance precursor ions that are selected for dissociation and the product ions are analyzed in a MS/MS scan mode. Thus, the precursor ions are not predetermined (MRM) but, rather, detected during the survey scan.
Generally described, data-dependent acquisition, which is also referred to, in various commercial implementations, as Information Dependent Acquisition (IDA), Data Directed Analysis (DDA), intelligent SRM (iSRM) and AUTO MS/MS, involves using data derived from an experimentally-acquired mass spectrum in an “on-the-fly” or “real-time” manner to direct the subsequent operation of a mass spectrometer. Utilization of data-dependent acquisition methods in a mass spectrometer provides the ability to make automated, real-time decisions in order to maximize the useful information content of the acquired data, thereby avoiding or reducing the need to perform multiple chromatographic runs or injections of the analyte sample. These methods can be tailored for specific desired objectives, such as enhancing the number of peptide identifications from the analysis of a complex mixture of peptides derived from a biological sample.
Data-dependent acquisition methods may be characterized as having one or more input criteria, and one or more output actions. The input criteria employed for conventional data-dependent methods are generally based on parameters such as intensity, intensity pattern, mass window, mass difference (neutral loss), mass-to-charge (m/z) inclusion and exclusion lists, and product ion mass. The input criteria are employed to select one or more ion species that satisfy the criteria. The selected ion species are then subjected to an output action (examples of which include performing MS/MS or MSn analysis and/or high-resolution scanning). In one instance of a typical data-dependent experiment, a group of ions is mass analyzed, and ion species having mass spectral intensities exceeding a specified threshold are subsequently selected as precursor ions for MS/MS analysis, which may involve operations of isolation, dissociation of the precursor ions, and mass analysis of the product ions.
Many mass spectrometer systems employ an ion mobility apparatus between an ion source and the mass spectrometer apparatus in order to selectively filter the ions prior to mass spectrometric analysis. In ion mobility spectrometry devices, separation of gas-phase ions is accomplished by exploiting variations in ion drift velocities under an applied electric field arising from differences in ion mobility. One well-known type of ion mobility spectrometry device is the High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) cell, also known by the term Differential Ion Mobility Spectrometry (DMS) cell, which separates ions on the basis of a difference in the mobility of an ion at high field strength (commonly denoted as Kh) relative to the mobility of the ion at low field strength (commonly denoted as K).
U.S. Pat. No. 6,504,149, in the name of inventors Guevremont and Purves, teaches the coupling of a FAIMS apparatus to a mass spectrometer. Briefly described, a FAIMS cell comprises a pair of spaced apart electrodes that define therebetween a separation region through which a stream of ions is directed. An asymmetric oscillatory voltage waveform comprising a high voltage component and a lower voltage component of opposite polarity, together with a non-oscillatory DC voltage (referred to as the compensation voltage, or CV) is applied to one of the electrodes. When the ion stream contains several species of ions, generally only one ion species is selectively or preferentially transmitted through the FAIMS cell for a given combination of asymmetric waveform peak voltage (referred to as the dispersion voltage, or DV) and CV. The remaining species of ions drift toward one of the electrode surfaces and are neutralized. The FAIMS cell may be operated in single ion detection mode, wherein the DV and CV are maintained at constant values, or alternatively the applied CV may be scanned with time to sequentially transmit ion species having different mobilities. FAIMS cells may be used for a variety of purposes, including providing separation or filtering of an ion stream prior to entry into a mass analyzer. When used as a pre-filter for a mass spectrometer, the FAIMS apparatus provides a way of eliminating isobaric interference ions which might accidentally have a mass-to-charge ratio nearly identical to that of an analyte of interest.
FIG. 1 schematically depicts a first known system 100 for analyzing ions that includes a FAIMS device 155 coupled to a mass spectrometer 157. The known FAIMS device 155 illustrated in FIG. 1 is an example of a type of device that has been referred to as a “side-to-side FAIMS” or a “perpendicular-gas-flow-FAIMS” (e.g., see U.S. Pat. No. 6,713,758 and international application publication No. WO01/69216). A solution of sample to be analyzed is introduced as a spray of liquid droplets into an ionization chamber 105 via atmospheric pressure ion source 110. Ionization chamber 105 is maintained at a high pressure relative to the regions downstream in the ion path, typically at or near atmospheric pressure. Atmospheric pressure ion source 110 may be configured as an electrospray ionization (ESI) probe, wherein a high DC voltage (either positive or negative) is applied to the capillary or “needle” through which the sample solution flows. Other suitable ionization techniques may be utilized in place of ESI, including without limitation such well-known techniques as atmospheric pressure chemical ionization (APCI), heated electrospray ionization (HESI), and thermospray ionization.
Ions produced by the ion source enter the FAIMS cell 155 through an aperture 117 in an entrance plate 120 and then through an inlet orifice 150 after passing through an expansion chamber 111. The expansion chamber is provided with a gas, typically helium or other inert gas, which is introduced into the expansion chamber 111 via a gas conduit 113. A portion of the gas flows back into the ionization chamber 105 through entrance plate aperture 117 in counter-flow to the ions and droplets and serves to desolvate charged droplets. Another portion of the gas combines with the analyte ions in chamber 111 and serves as a carrier gas through the FAIMS cell 155. The combined ion/carrier gas flow then enters FAIMS cell 155 through inlet orifice 150. The carrier gas flow may be carefully metered to maintain flow rates within predetermined limits which will depend on the FAIMS cell size, electrode geometry, and operational considerations. An electrical potential difference is maintained between the entrance plate 120 and the FAIMS cell 155 and, thus, physical separation is maintained between these components. Accordingly, a non-conducting sealing element 173, such as a gasket or O-ring maintains the FAIMS gas within the apparatus and prevents contamination of this gas from outside air. Because of drawing-space limitations, this sealing element is not explicitly shown in some of the accompanying drawings.
Generally speaking, the side-to-side FAIMS cell 155 includes inner and outer electrodes 165 and 170 having radially opposed surfaces, which define therebetween an annular separation region 175 (an “analytical gap”) through which the ions are transported. The side-to-side FAIMS cell geometry depicted in FIG. 1 as well as in other figures herein provides a configuration in which the longitudinal axes (axes of cylindrical surfaces, directed out of the page) of inner electrode 165 and outer electrode 170 are oriented transversely with respect to the overall direction of ion flow. The principles of the design and operation of FAIMS cells and other ion mobility spectrometry devices have been extensively described elsewhere in the art (see, for example, U.S. Pat. No. 6,639,212 to Guevremont et al., incorporated by reference herein in its entirety), and hence will not be described in detail herein. In brief, the carrier gas and ions flow through the separation region 175 from inlet orifice 150 to exit orifice 185. Ion separation is effected within the separation region (analytical gap) 175 of the FAIMS cell 155 by applying an asymmetric waveform having a peak voltage (DV) and a compensation voltage (CV) to one of the inner or outer electrodes, 165, 170. The values of CV and DV are set to allow transmission of a selected ion species through separation region 175. Other ion species having different relative values of high field and low field mobilities will migrate to the surface of one of the electrodes and will be neutralized.
Still referring to FIG. 1, the selected ions emerge from the FAIMS cell 155 through exit orifice 185 and pass through a small gap 183 separating the FAIMS cell 155 from a mass spectrometer 157. Whereas most of the carrier gas exhausts through the gap 183 at atmospheric pressure, ions are electrostatically guided into at least one reduced pressure chamber 188 of the mass spectrometer 157 through an orifice in the mass spectrometer or through an ion transfer tube 163. The at least one reduced pressure chamber may be evacuated by a vacuum port 191. At least a portion of ion transfer tube 163 may be surrounded by and in good thermal contact with a heat source, such as heater jacket 167. The heater jacket 167, which may take the form of a conventional resistance heater, is operable to raise the temperature of ion transfer tube 163 to promote further desolvation of droplets entering the ion transfer tube 163.
From the at least one reduced pressure chamber 188, ions are transferred through an orifice 193 of a skimmer 194 into a high vacuum chamber 195 maintained at a low pressure (typically around 100 millitorr) relative to the reduced pressure chamber 188. The high vacuum chamber 195 is typically evacuated by turbo or similar high-vacuum pumps via a vacuum port 197. The skimmer 194 may be fabricated from an electrically conductive material, and an offset voltage may be applied to skimmer 194 to assist in the transport of ions through interface region and into skimmer orifice 193. Ions passing through skimmer orifice 193 may be focused or guided through ion optical assembly 198, which may include various electrodes forming ion lenses, ion guides, ion gates, quadrupole or octopole rod sets, etc. The ion optical assembly 198 may serve to transport ions to an analyzer 199 for mass analysis. Analyzer 199 may be implemented as any one or a combination of conventional mass analyzers, including (without limitation) a quadrupole mass analyzer, ion trap, or time-of-flight analyzer.
The inlet orifice 150 of the conventional FAIMS apparatus 155 comprises a simple hole of circular cross section having a constant inner diameter. Recently, U.S. Pat. No. 8,664,593, which is assigned to the assignee of the instant invention, described side-to-side FAIMS apparatuses having curved ion inlet orifices which provide for more efficient transfer of analyte ions through the analytical gap. FIG. 2 shows the FAIMS gas flow into an electrode set that is provided with a so-modified ion inlet orifice. The modified ion inlet orifice acts to decrease the volume and rate of gas flow directly onto the inner electrode adjacent to the orifice, thus significantly reducing neutralization of analyte ions. Note that the inner electrode 165 is generally a right-circular cylindrical rod having an axis 177 that is parallel to the length of the rod (see FIG. 5A). In the cross sectional representations of FIG. 2 and FIG. 3, the axis 177 is perpendicular to the plane of the drawing and is thus indicated as a piercing point (“+” symbol).
The FAIMS apparatus 109 that is schematically illustrated in FIG. 2 is generally similar to the FAIMS apparatus 155 shown in FIG. 1 except with regard to the shape of the ion inlet orifice. Inset 30 of FIG. 2 illustrates an enlarged view of the vicinity of the ion inlet orifice 151 of the FAIMS 109. The walls 31 of the ion inlet orifice 151 of the FAIMS apparatus 109 are convexly curved between the orifice inlet end 32 and the orifice outlet end 33. Thus, the inner diameter of the ion inlet orifice is at a minimum value within the orifice. Because of the curvature, the inner diameter of the ion inlet orifice 151 smoothly increases or flares outward in both directions (i.e., towards the two ends of the orifice or, equivalently, towards and away from the inner electrode 177) away from the region of minimum diameter. The gas flow in the vicinity of the rounded walls of the ion inlet orifice 151, as determined by fluid dynamic calculations, demonstrates the so-called Coand{hacek over (a)} effect, which is the general tendency of a fluid jet to be drawn towards and follow the contour of a curved solid surface. By means of the Coand{hacek over (a)} effect, the carrier gas flow entering the analytical gap 175 of the FAIMS apparatus 109 (FIG. 2) is kept closer to the curvature of the entrance orifice than would otherwise be the case. This behavior allows for incorporation of the gas stream into the gap and away from the inner electrode as is indicated in FIG. 2 by the smooth divergence of gas flow vectors away from the center electrode 165 and into the analytical gap 175. The smooth divergence of the carrier gas into away from the center electrode and into the analytical gap 175 is expected to urge ions along similar pathways, thereby reducing the proportion of ions that are lost as a result of collision with the center electrode and improving ion transmission through the FAIMS apparatus. As indicated by the fluid dynamics calculations, the smooth divergence also leads to a larger zone of laminar flow within the analytical gap, with reduced recirculation flow near the entrance orifice.
FIG. 3 shows the results of combined fluid dynamic and ion trajectory modeling, through the FAIMS apparatus 109. FIG. 4 shows a comparison between the transmission efficiency (curve 42) of the apparatus having the curved an inlet orifice 151 and shown in FIG. 3 with that of the prior FAIMS apparatus shown in FIG. 1 (curve 44). It is evident from the shape of the ion cloud 129 in FIG. 3 that the curved orifice design promotes a smooth bifurcation of ion flow prior around the center electrode 165 and into the analytical gap 175. The smooth flow bifurcation appears to have the effect of reducing gas recirculation flow with the analytical gap just after passing through the inlet orifice, thereby significantly reducing ion neutralization at both inner and outer electrodes.
The simple re-design of the cross-sectional shape of the ion inlet orifice as described above improves the uniformity of flow of carrier gas through the FAIMS apparatus. This smoother flow is such that there is highly reduced flow rate of the carrier gas (and entrained ions) directly onto the electrodes, relative to the conventional FAIMS apparatus 155 (FIG. 1). This smoother flow is believed to have a major effect in yielding the results of FIG. 4, in which simulated CV scans through the FAIMS 109 and through the FAIMS 155 are shown as curves 42 and 44, respectively.
One of the limiting characteristics of conventional FAIMS apparatuses that have precluded them from being used on a mass spectrometer employing an LC/MS/MS workflow or other varieties of data-dependent acquisition has been the long transit time or residence time of ions through the FAIMS analyzer. The residence time required for ions to transit through the FAIMS analyzer gap can range between 50-100 ms. In general, there does not exist a simple one-to-one correspondence between differential ion mobility and ion m/z ratio. Thus, a mass analyzer that receives ions from a FAIMS apparatus should remain set to detect only the m/z value of a particular analyte ion species of interest during the entire time that the FAIMS is operated so as to transmit ions having the particular differential ion mobility associated with that particular analyte ion species. If the mass analyzer were to be set to detect a different m/z ratio during this time, generally no ions would be detected, since the FAIMS would generally eliminate all other ion species, based on their various values of differential ion mobility.
In accordance with the above considerations, the FAIMS residence time thus defines the period that the first mass analyzer (Q1) must spend on a single mass-to-charge ratio (m/z) isolation. As discussed above, Q1 only requires, at most, a few tens of milliseconds, in the absence of a FAIMS pre-filter, to isolate or filter ions comprising a single m/z range, thus permitting analysis of up to ˜100 or more isolations per second. However, increasing the Q1 dwell time in order to match the 50-100 ms residence time of the conventional FAIMS apparatus may result in insufficient number of scans to define a stable chromatographic peak structure. This limitation is often reached when employing a FAIMS apparatus coupled to a mass spectrometer. Generally, the time between scans or the cycle time will be the dwell time plus the inter-scan-delay-time plus the ion residence time. Given the time requirements of the conventional FAIMS apparatus, the cycle time required to perform at least an MS scan for each one of the 100 precursors in the MRM list is approximately 10.2 seconds (as opposed to 1.2 seconds per cycle in the absence of the FAIMS). Thus, only one MS scan per precursor ion can be made across a 12-second wide LC peak.
Another limiting characteristic of conventional FAIMS that has precluded its large-scale use with an LC/MS/MS workflow or other data-dependent acquisition workflow has been the generally low ion throughput transmission of conventional FAIMS apparatuses (e.g., see curve 44 of FIG. 4). The throughput remains low for all ions even when the FAIMS apparatus is employed as a passive transmission device by ceasing to apply the FAIMS dispersion voltage (DV) and compensation voltage (CV), thereby changing its operating mode to “non-dispersive” whereby the FAIMS apparatus acts as a passive device that non-selectively transmits all ions. The low throughput associated with conventional FAIMS apparatuses can lead to an unacceptable Limit of Detection (LOD) of detection and limit of quantitation (LOQ) for assays performed by the mass spectrometer to which the FAIMS apparatus is coupled.
A prior solution to these limitations associated with the use of a FAIMS apparatus as a pre-filter for a mass spectrometer has been to either physically remove an existing FAIMS apparatus from a mass spectrometer that is to perform data-dependent acquisition or to employ, for data-dependent acquisition, a separate mass spectrometer that is not coupled to a FAIMS apparatus. Clearly, such measures are neither time-effective nor cost-effective. Thus, there is a need in the art to be able to realize the filtering advantages of FAIMS during an LC-FAIMS-MRM analysis while also maintaining an adequate number of mass spectral samples for precursor/product ion pair of interest. In a related fashion, there is a need in the art to be able to realize the filtering advantages of FAIMS during the execution of general mass spectral data-dependent acquisition analyses while also maintaining adequate mass spectral limits of detection and quantitation. The present invention addresses these needs.